This invention relates to nucleic acid molecules and methods for sequence-specific methylation of ribonucleic acid.
Ribosomes are large, complex particles that play a central role in protein synthesis. Ribosomes are produced in a discrete region of the cell nucleus, designated the nucleolus. Eukaryotic ribosomes consist of 40S and 60S subunits, which each contain multiple ribonucleic acid (RNA) and protein components. For example, the 40S ribosomal subunit contains 18S ribosomal RNA (rRNA) and the 60S ribosomal subunit contains 5S, 28S (28S in metazoans and 25S in unicellular eukaryotes, such as yeast), and 5.8S rRNAs. Each of the rRNAs, except 5S rRNA, are transcribed in the nucleolus as segments of a large precursor RNA, which is cleaved in the production of the mature rRNAs. Additional rRNA processing events include modification of certain bases by, e.g., conversion of uridine to pseudo-uridine or methylation of ribose 2'-hydroxyl groups.
In addition to rRNAs, eukaryotic nucleoli contain complex populations of small nucleolar RNAs (snoRNAs), several of which have been shown to be required for rRNA processing (Maxwell et al., Annu. Rev. Biochem. 35:899, 1995; Filipowicz et al., Mol. Biol. Rep. 18:235, 1993; Fournier et al., Trends Biochem. Sci. 18:131, 1993; Sollner-Webb, Cell 75:403, 1993). snoRNAs associate with multiple proteins to form small nucleolar ribonucleoprotein (snoRNP) particles. For example, nearly all snoRNAs are associated with the nucleolar protein fibrillarin, which itself is required for normal processing and methylation of rRNA.
Some snoRNAs are encoded within the introns of genes, e.g., genes coding for proteins involved in ribosome synthesis and function. These snoRNAs contain conserved sequence motifs, and several of them also contain regions of sequence complementarity to conserved regions of rRNA of up to 21 nucleotides. In several cases, methylated nucleotides are present in the rRNA sequence elements that are complementary to these snoRNAs (Bachellerie et al., Trends Biochem. Sci. 20:261, 1995).